DIN EN 15890-2010 Foodstuffs - Determination of patulin in fruit juice and fruit based purée for infants and young children - HPLC method with liquid liquid partition cleanup and s.pdf

1、December 2010 Translation by DIN-Sprachendienst.English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).I

2、CS 67.080.10; 67.160.20; 67.230!$lYY“1735454www.din.deDDIN EN 15890Foodstuffs Determination of patulin in fruit juice and fruit based pure for infantsand young children HPLC method with liquid/liquid partition cleanup and solid phaseextraction and UV detectionEnglish translation of DIN EN 15890:2010

3、12Lebensmittel Bestimmung von Patulin in Fruchtsaft und Obstbrei fr Suglinge und Kleinkinder HPLC-Verfahren mit Reinigung durch Flssig/Flssig-Verteilung, Festphasenextraktionund UV-DetektionEnglische bersetzung von DIN EN 15890:2010-12Denres alimentaires Dosage de la patuline dans le jus de fruits

4、et la compote de fruits en alimentationinfantile Mthode par CLHP avec purification par partition liquide-liquide et extraction en phasesolide et dtection UVTraduction anglaise de DIN EN 15890:2010-12www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considere

5、d authoritative.11.10 18DIN EN 15890:2010-12 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 5 “Biotoxins”. The res

6、ponsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. Mycotoxins are extremely toxic secondary metabolites produced by fungi. Food that

7、is grown, harvested or stored under humid conditions can be infected by fungi whose metabolites invade the food. Because some mycotoxins are extremely toxic for humans, their reliable detection is especially important for consumer health and protection. In Germany the regulation Mykotoxin-Hchstmenge

8、nverordnung governs the maximum allowable content of mycotoxins in food. Since 2004 this regulation has contained specifications not only for aflatoxins, but also for ochratoxin A, fumonisins, deoxynivalenol (DON), and zearalenone. National regulations have been supplemented by EU-wide regulations r

9、egarding the maximum content of contaminants in food since 2001. The maximum content of mycotoxins in certain foods is also laid down in various other regulations. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 Nation

10、al Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15890 September 2010 ICS 67.080.10; 67.160.20; 67.230 English Version Foodstuffs - Determination of patulin in fruit juice an

11、d fruit based pure for infants and young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection Denres alimentaires - Dosage de la patuline dans le jus de fruits et la compote de fruits en alimentation infantile - Mthode par CLHP avec purification par

12、 partition liquide-liquide et extraction en phase solide et dtection UV Lebensmittel - Bestimmung von Patulin in Fruchtsaft und Obstbrei fr Suglinge und Kleinkinder - HPLC-Verfahren mit Reinigung durch Flssig/Flssig-Verteilung, Festphasenextraktion und UV-Detektion This European Standard was approve

13、d by CEN on 28 August 2010. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national st

14、andards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notifie

15、d to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembou

16、rg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights

17、of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15890:2010: EEN 15890:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .66 Procedure .87 HPLC analysis 98 Calculation . 109 Precision 1010 Te

18、st report . 11Annex A (informative) Typical chromatograms 12Annex B (informative) Precision data . 14Bibliography . 16DIN EN 15890:2010-12 EN 15890:2010 (E) 3 Foreword This document (EN 15890:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretar

19、iat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to th

20、e possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free

21、Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungar

22、y, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not

23、purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. DIN EN 15890:2010-12 EN 15890:2010 (E) 4 1 Scope This

24、 European Standard specifies a method for the determination of patulin in fruit juices and fruit-based pure, such as baby food pure, using high performance liquid chromatography with ultra-violet detection (HPLC-UV). Using naturally contaminated and spiked samples this method has been validated for

25、the determination of patulin in apple juice, at levels ranging from 3,0 g/kg to 15,5 g/kg, and in fruit-based baby food pure, at levels ranging from 3,4 g/kg to 17,9 g/kg. Baby food fruit pure used in this study contained a mixture of the following ingredients which are commercially available on the

26、 European market: blueberry; apple; banana; lemon; wheat biscuits; wheat syrup; whole milk; and vegetable oil. A detailed listing, including the fractions, of each product used in this study is given in 1. Further information on validation, see Clause 9 and Annex B. 2 Normative references The follow

27、ing referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Speci

28、fication and test methods (ISO 3696:1987) 3 Principle Patulin is extracted from apple juice, or fruit-based pure, with a mixture of ethyl-acetate and hexane in the presence of sodium sulfate and sodium hydrogen carbonate. An aliquot of the extract is purified by solid-phase extraction and evaporated

29、 The residue is re-dissolved in water of pH = 4 and patulin is separated by reverse phase (RP)-HPLC and quantitatively determined by UV detection. 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified

30、 Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to the reagents listed may be used. 4.2 Perchloric acid, the mass fraction w(HClO4) 60 % in water. 4.3 Sand, 50 mesh to 70 mesh particle size. 4.4 Silicagel solid

31、 phase extraction (SPE) cartridges (500 mg SiO2). 4.5 Sodium sulfate anhydrous, Na2SO4. 4.6 Sodium hydrogen carbonate, NaHCO3. 4.7 Glacial acetic acid, w(CH3COOH) 98 % in water. DIN EN 15890:2010-12 EN 15890:2010 (E) 5 4.8 Water of pH = 4. Adjust water to pH = 4 with glacial acetic acid (4.7). 4.9 A

32、bsolute ethanol, w(CH3CH2OH) 99,7 % in water. 4.10 Acetonitrile. WARNING Acetonitrile is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.11 Ethyl acetate. 4.12 n-Hexane

33、 4.13 Extraction solvent. Add 60 ml of ethyl acetate (4.11) to 40 ml of n-hexane (4.12). 4.14 Mixture of glacial acetic acid and ethyl acetate. Add 3 ml of glacial acetic acid (4.7) to 97 ml of ethyl acetate (4.11). 4.15 HPLC mobile phase. Mix 990 parts per volume of water with up to ten parts per

34、volume of acetonitrile (4.10) and one part per volume of perchloric acid (4.2). The amount of acetonitrile will depend upon the type of samples analysed and their characteristic pattern of interferences after clean-up (see Annex A for typical chromatograms) and the HPLC column chosen for analysis. D

35、egas this solution before use. NOTE A mobile phase of 990 part of water with one part of perchloric acid has been found to give sufficient separation between patulin and other interfering substances (in particular 5-hydroxymethylfurfural when used in combination with a Synergy1)column of 250 mm leng

36、th and 4,6 mm diameter with a particle size of 4 m and 8 nm porosity (see 5.13.4). 4.16 Patulin. WARNING Patulin is a suspect mutagen and has been reported to have immunotoxic and neurotoxic properties. Gloves and safety glasses should be worn at all times and all standard and sample preparation sta

37、ges should be carried out in a fume cupboard. 4.17 Patulin stock solution. Dissolve 5 mg of patulin or the contents of one ampoule (if patulin has been obtained as a film) in ethyl acetate (4.11). Transfer the solution to a 25 ml volumetric flask and dilute to volume with ethyl acetate to produce a

38、solution containing approximately 200 g/ml of patulin. 1) Synergyis a trade name of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may

39、be used if they can be shown to lead to the same results. DIN EN 15890:2010-12 EN 15890:2010 (E) 6 Store this solution in a freezer at approximately - 18 C. Confirm the mass concentration of the solution if it is older than six weeks. Ensure the solution is allowed to reach room temperature before u

40、se to avoid incorporation of water by condensation. 4.18 Patulin standard solution. Evaporate 1 000 l of the stock solution (4.17) to dryness under nitrogen and then immediately dissolve it in 20 ml of ethanol (4.9) to obtain a mass concentration of approximately 10 g/ml of patulin. To determine the

41、 exact mass concentration, record the absorption curve between 250 nm and 350 nm in a 1 cm quartz cell with ethanol as reference. Identify the wavelength for maximum absorption. Calculate the mass concentration of patulin, pat, in micrograms per millilitre, using Equation 1: bMA=100maxpat(1) where A

42、max is the absorption determined at the maximum of the absorption curve (here: at approximately 276 nm); M is the molar mass, in grams per mole, of patulin (M = 154 g/mol); is the molar absorption coefficient, in square metres per mole, of patulin in ethanol (here: 1 460 m2/mol, see 2); b is the opt

43、ical path length, in centimetres, of the quartz cell. Store this solution in a freezer at approximately - 18 C. A solution stored in this way is stable for several months. Ensure that the standard solution is allowed to reach room temperature before use to avoid incorporation of water by condensatio

44、n. Confirm the concentration of the solution if it is older than six weeks. 4.19 Spiking solutions. For spiking experiments at levels of 10 ng/ml and 25 ng/ml patulin in the sample, prepare spiking solutions of patulin in water of pH = 4 (4.8) at mass concentrations of 200 ng/ml and 500 ng/ml, respe

45、ctively. These solutions can be obtained by evaporating exactly 100 l and 250 l respectively of the stock solution (4.17) to dryness under nitrogen in a 100 ml volumetric flask, followed by immediate dissolution in water of pH = 4 (4.8) to obtain a mass concentration of approximately 200 ng/ml and 5

46、00 ng/ml respectively of patulin, depending on the exact mass concentration of patulin in the stock solution. Make sure that the patulin is completely dissolved in the water of pH = 4 before the volumetric flask is filled up to the mark. In case the patulin standard solution (4.18) has a different m

47、ass concentration than 10 g/ml, adjust spiking solutions by calculating the correct aliquots in order to take account of the actual mass concentration of the standard solution determined in 4.18. Store this solution in a refrigerator at 4 C. A solution stored in this way is stable for at least eight

48、 weeks. 5 Apparatus 5.1 General Usual laboratory apparatus and, in particular, the following. DIN EN 15890:2010-12 EN 15890:2010 (E) 7 5.2 Displacement pipettes, of e.g. 5 ml, 1 ml , 200 l and 50 l capacity with appropriate pipette tips. 5.3 Analytical balance, capable of weighing to 0,1 mg. 5.4 UV

49、spectrometer, double beam and recording suitable for measurement at 250 nm to 350 nm. 5.5 Quartz cells, with an optical path length of 1 cm. 5.6 Centrifuge, capable of operating at 400 g. 5.7 Centrifuge tubes, of 25 ml capacity with screw cap lids. 5.8 Mechanical shaker. 5.9 Evaporation block, capable of maintaining a temperature of 40 C, with nitrogen supply. 5.10 Glass vial, of 6 ml capacity with screw cap. 5.11 Syringe, gas tight with a polytetrafluoroe